Ionotropic glutamate receptors (iGluRs) are glutamate-activated, cation-selective ion channels that mediate the majority of millisecond-time scale signaling in the nervous system. The iGluR family of receptors is composed of three major subtypes, the AMPA, NMDA and kainate receptors, each of which plays a crucial role in nervous system development and function. The disruption of normal glutamatergic signaling underpins multiple neurological disorders, including Alzheimer?s disease, depression, schizophrenia and epilepsy. Accordingly, iGluRs are the targets for numerous therapeutic agents to treat seizure and mood disorders, as well as schizophrenia. Several immunological disorders of the nervous system also involve iGluRs, with the most prevalent being NMDA receptor encephalitis, a severe autoimmune disease for which there are few promising treatments. Despite the widespread distribution of iGluRs throughout the human nervous system, and even though they are involved in many debilitating neurological diseases and disorders, the molecular mechanisms of iGluR function are not yet fully elucidated. Moreover, iGluRs are not isolated at their sites of function, typically the synapses, but rather are found in complexes with multiple auxiliary proteins. Indeed, the organization and molecular function of these auxiliary proteins also remains to be fully elucidated. Thus, the major focus of the research proposed in this grant application is to elucidate the molecular structure and mechanisms of function for AMPA and NMDA receptors, alone and in complexes with key auxiliary proteins. Here we will develop methods to isolate native AMPA receptors from whole brain and from the hippocampus and cerebellum, as complexes with their auxiliary proteins. We will then elucidate the structures of these receptor complexes by single particle cryo-EM, using subunit specific antibody fragments to tag subunits. With the structures in hand, we will design mutants to test important aspects of structure and mechanisms, carrying out biochemical and electrophysiological experiments to probe receptor function. In parallel, we will also study the NMDA receptors, here focusing on a structure-based mechanism for receptor gating, investigating the structure of the GluN1/GluN2A receptor in complexes with the agonists glycine and glutamate, as well as with allosteric modulators and ion channel blockers. Through these studies we aim to answer the question of how neurotransmitters activate the receptor and how allosteric modulators increase or decrease receptor activity. In parallel with AMPA receptor studies, we will also isolate native GluN1-containing NMDA receptors from rodent brain tissue, with the aim being to elucidate the first structure of native NMDA receptors, mapping the positions of the GluN1 and GluN2 subunits in the receptor complex. Taken together, our studies will define structure-based, biochemical mechanisms for iGluR function and they will lay the foundation for development of new therapeutic agents.